Second Harmonic Generation analysis of the ECM in idiopathic pulmonary fibrosis Idiopathic pulmonary fibrosis (IPF) is a disorder characterized by unrelenting scarring and stiffening of the lungs that leads to the death of an estimated 34,000 individuals in the U.S. each year. Unfortunately, individuals with IPF have extremely limited treatment options, as no effective drugs have been identified to halt the progression of fibrosis. Despite the importance of collagens to the structural organization both normal and remodeled ECM, little is known about how collagen structure in IPF differs from that of normal tissue architecture. There is a clear need to develop highly specific/sensitive techniques to probe collagen structure and organization in IPF tissues. In this project we will implement new collagen specific analyses using the high resolution microscopy technique of Second Harmonic Generation (SHG). This method is sensitive to both the fibrillar organization and also sub-resolution aspects of macro and supramolecular assembly. Here we will utilize SHG microscopy to: 1) determine the how pathologic collagen organization (seen in IPF) differs from normal tissue; 2) identify and quantify areas of active fibrosis (enriched in collagen III) from old or mature fibrosis (high in collagen I) in IPF lung specimens; 3) assess changes in elastin and collagen distribution during disease progression; and 4) correlate areas of high collagen III/I signal in IPF histologic samples with clinical markers of disease activity. As part of the project, we will develop customized automated machine vision routines to automatically classify tissues in terms of severity. We will specifically focus all of our efforts on studying structure around fibroblastic foci, which will be identified by other microscope modalities. These foci are thought to be at the leading edge of ECM remodeling but the dynamics of their formation in relationship to the overall fibrotic process remain unclear. We hypothesize that these structural changes will serve as label- free biomarkers of IPF and further hypothesize that the collagen is altered specifically around foci in a manner which is associated with disease progression. The information gained may form the basis of future prognostic/diagnostic schemes. We propose 2 Aims: Aim 1 Polarization resolved SHG to determine distribution of Col I/III and other ECM changes in different stages of IPF. Aim 2. Develop classification system of morphological changes in IPF visualized by SHG.